Although the existing agents for treating cancer have shown promising results, the inherent genetic plasticity of the cancer cells continues to undermine the efficacy of these treatments. One of the possible solutions to address this problem is to attack the basic machinery that helps the cancer cells and tumors to adapt successfully under stress. Heat shock protein 90 (Hsp90) is the major molecular chaperone that helps its client proteins to overcome this stress by assisting general protein folding and preventing non-specific aggregation. By directly targeting Hsp90 one can, therefore, shut down the activities associated with cancer, at the same time stepping around the multiple signaling pathways. Inhibitors of the cytosolic Hsp90 in clinical studies have revealed that while once-a-week administration is tolerated, hepato-toxicity develops after several days of daily administration. If dose-limiting toxicity of these existing compounds is structure-related, there is a need for better compounds that are structurally diverse and can overcome the observed toxicity. Recent studies have shown that a close analog of cytosolic Hsp90 is present in the mitochondria, known as TNF Receptor-Associated Protein 1 (TRAP1). Reports have implicated TRAP1 in protecting cells from mitochondria-mediated apoptosis by oxidative stress. Strategies aimed at inhibiting TRAP1, based on novel TRAP1 ATPase antagonists, induce sudden collapse of mitochondrion function and apoptosis, thereby improving the efficacy of anticancer treatments. Therefore, from this perspective, targeting the TRAP1/Hsp90 chaperones together may represent a novel double-pronged molecular mechanism to overcome the limitations of the existing cancer therapies including drug resistance. Therefore, in the current proposal, we would like to take a combinatorial approach to selectively identify inhibitors of Hsp90 and TRAP1 that are sub-cellular specific and/or inhibitors with dual activity simultaneously targeting the N-terminal domains of cytosolic Hsp90 and mitochondria TRAP1. We will use our proprietary differential fragment-based screening combined with "dial in-dial out" structure based approach to generate effective anticancer drugs with a novel mechanism of action. Given the high degree of sequence homology (~70%) between the N-terminal domains of Hsp90 and TRAP1 (Fig. 3), we would like to employ this approach to discover selective and dual- active inhibitors and develop them into effective anticancer drugs. PUBLIC HEALTH RELEVANCE: The differential fragment-based screening technology combined with biochemical and medicinal chemistry approaches being used in this proposal will expedite the discovery and development of novel, safe and potent anti-cancer and neurodegenerative drug candidates with novel mechanism of action targeting two key proteins Hsp90 and TRAP1.